Proteases from gram-positive organisms

ABSTRACT

The present invention relates to the identification of novel cysteine proteases in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for the  Bacillus subtilis  cysteine proteases CP1, CP2 and CP3. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding CP1, CP2 or CP3. The present invention also provides host cells further comprising nucleic acid encoding desired heterologous proteins such as enzymes. The present invention also provides a cleaning composition comprising a cysteine protease of the present invention.

FIELD OF THE INVENTION

[0001] The present invention relates to cysteine proteases derived fromgram-positive microorganisms. The present invention provides nucleicacid and amino acid sequences of cysteine protease 1, 2 and 3 identifiedin Bacillus. The present invention also provides methods for theproduction of cysteine protease 1, 2 and 3 in host cells as well as theproduction of heterologous proteins in a host cell having a mutation ordeletion of part or all of at least one of the cysteine proteases of thepresent invention.

BACKGROUND OF THE INVENTION

[0002] Gram-positive microorganisms, such as members of the groupBacillus, have been used for large-scale industrial fermentation due, inpart, to their ability to secrete their fermentation products into theculture media. In gram-positive bacteria, secreted proteins are exportedacross a cell membrane and a cell wall, and then are subsequentlyreleased into the external media usually maintaining their nativeconformation.

[0003] Various gram-positive microorganisms are known to secreteextracellular and/or intracellular protease at some stage in their lifecycles. Many proteases are produced in large quantities for industrialpurposes. A negative aspect of the presence of proteases ingram-positive organisms is their contribution to the overall degradationof secreted heterologous or foreign proteins.

[0004] The classification of proteases found in microorganisms is basedon their catalytic mechanism which results in four groups: the serineproteases; metalloproteases; cysteine proteases; and aspartic proteases.These categories can be distinguished by their sensitivity to variousinhibitors. For example, the serine proteases are inhibited byphenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate(DIFP); the metalloproteases by chelating agents; the cysteine enzymesby iodoacetamide and heavy metals and the aspartic proteases bypepstatin. The serine proteases have alkaline pH optima, themetalloproteases are optimally active around neutrality, and thecysteine and aspartic enzymes have acidic pH optima (BiotechnologyHandbooks, Bacillus. vol. 2, edited by Harwood, 1989 Plenum Press,N.Y.).

[0005] The activity of cysteine protease depends on a catalytic dyad ofcysteine and histidine with the order differing among families. The bestknown family of cysteine proteases is that of papain having catalyticresidues Cys-25 and His-159. Cysteine proteases of the papain familycatalyze the hydrolysis of peptide, amide, ester, thiol ester and thionoester bonds. Naturally occurring inhibitors of cysteine proteases of thepapain family are those of the cystatin family (Methods in Enzymology,vol. 244, Academic Press, Inc. 1994).

SUMMARY OF THE INVENTION

[0006] The present invention relates to the unexpected and surprisingdiscovery of three heretofore unknown or unrecognized cysteine proteasesfound in Bacillus subtilis, designated herein as CP1, CP2 and CP3,having the nucleic acid and amino acid as shown in FIGS. 1A-1B, FIGS.5A-5B and 6A-68, respectively. The present invention is based, in part,upon the presence of the characteristic cysteine protease amino acidmotif GXCWAF found in uncharacterised translated genomic nucleic acidsequences of Bacillus subtilis. The present invention is also based inpart upon the structural relatedness that CP1 has with the cysteineprotease papain specifically with respect to the location of thecatalytic histidine/alanine and asparagine/serine residues and thestructural relatedness that CP1 has with CP2 and CP3.

[0007] The present invention provides isolated polynucleotide and aminoacid sequences for CP1, CP2 and CP3. Due to the degeneracy of thegenetic code, the present invention encompasses any nucleic acidsequence that encodes the CP1, CP2 and CP3 amino acid sequence shown inthe Figures.

[0008] The present invention encompasses amino acid variations ofB.subtilis CP1, CP2 and CP3 amino acids disclosed herein that haveproteolytic activity. B. subtilis CP1, CP2 and CP3, as well asproteolytically active amino acid variations thereof, have applicationin cleaning compositions. In one aspect of the present invention, CP1,CP2 or CP3 obtainable from a gram-positive microorganism is produced onan industrial fermentation scale in a microbial host expression system.In another aspect, isolated and purified recombinant CP1, CP2 or CP3obtainable from a gram-positive microorganism is used in compositions ofmatter intended for cleaning purposes, such as detergents. Accordingly,the present invention provides a cleaning composition comprising atleast one of CP1, CP2 and CP3 obtainable from a gram-positivemicroorganism. The cysteine protease may be used alone in the cleaningcomposition or in combination with other enzymes and/or mediators orenhancers.

[0009] The production of desired heterologous proteins or polypeptidesin gram-positive microorganisms may be hindered by the presence of oneor more proteases which degrade the produced heterologous protein orpolypeptide. Therefore, the present invention also encompassesgram-positive microorganism having a mutation or deletion of part or allof the gene encoding CP1 and/or CP2 and/or CP3, which results in theinactivation of the CP1 and/or CP2 and/or CP3 proteolytic activity,either alone or in combination with deletions or mutations in otherproteases, such as apr, npr, epr, mpr for example, or other proteasesknown to those of skill in the art. In one embodiment of the presentinvention, the gram-positive organism is a member of the genus Bacillus.In another embodiment, the Bacillus is Bacillus subtilis.

[0010] In another aspect, the gram-positive microorganism host havingone or more deletions or mutations in a cysteine protease of the presentinvention is further genetically engineered to produce a desiredprotein. In one embodiment of the present invention, the desired proteinis heterologous to the gram-positive host cell. In another embodiment,the desired protein is homologous to the host cell. The presentinvention encompasses a gram-positive host cell having a deletion orinterruption of the naturally occurring nucleic acid encoding thehomologous protein, such as a protease, and having nucleic acid encodingthe homologous protein or a variant thereof re-introduced in arecombinant form. In another embodiment, the host cell produces thehomologous protein. Accordingly, the present invention also providesmethods and expression systems for reducing degradation of heterologousor homologous proteins produced in gram-positive microorganismscomprising the steps of obtaining a Bacillus host cell comprisingnucleic acid encoding said heterologous protein wherein said host cellcontains a mutation or deletion in at least one of the genes encodingcysteine protease 1, cysteine protease 2 and cysteine protease 3; andgrowing said Bacillus host cell under conditions suitable for theexpression of said heterologous protein. The gram-positive microorganismmay be normally sporulating or non-sporulating.

[0011] The present invention provides methods for detecting grampositive microorganism homologs of B. subtilis CP1, CP2 and CP3 thatcomprises hybridizing part or all of the nucleic acid encoding B.subtilis CP1, CP2 and CP3 with nucleic acid derived from gram-positiveorganisms, either of genomic or cDNA origin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A-1B shows the DNA (SEQ ID NO:1) and amino acid sequencefor CP1 (YJDE) (SEQ ID NO:2).

[0013]FIG. 2 shows an amino acid alignment with papain (SEQ ID NO:3)(accession number papa_carpa.p) with the cysteine protease CP1,designated YJDE. For FIGS. 2, 3 and 4, the motif GXCWAF has been markedalong with the catalytic cysteine and the conserved catalytichistidine/alanine and asparagine/serine residues.

[0014]FIG. 3 shows amino acid alignment of CP1 (YJDE) (SEQ ID NO:2) withCP3 (PMI) (SEQ ID NO:5).

[0015]FIG. 4 shows the amino acid alignment of CP1 (YJDE) (SEQ ID NO:2)with CP2 (YdhS).

[0016] FIGS. 5A-5B shows the amino acid (SEQ ID NO:6) and nucleic acidsequence for CP2 (YdhS) (SEQ ID NO:7).

[0017] FIGS. 6A-6B shows the amino acid (SEQ ID NO:4) and nucleic acidsequence for CP3 (PMI) (SEQ ID NO:5).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Definitions

[0019] As used herein, the genus Bacillus includes all members known tothose of skill in the art, including but not limited to B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautusand B. thuringiensis.

[0020] The present invention relates to novel CP1, CP2 and CP3 from grampositive organisms. In a preferred embodiment, the gram-positiveorganisms is a Bacillus. In another preferred embodiment, thegram-positive organism is Bacillus subtilis. As used herein, “B.subtilisCP1, CP2 or CP3 ” refers to the amino acid sequences shown in Figures.FIGS. 1A-1B show the amino acid and nucleic acid seqeunce for CP1(YJDE); FIGS. 5A-5B show the amino acid and nucleic acid sequence forCP2 (YDHS); and FIGS. 6A-6B show the amino acid and nucleic acidsequences for CP3 (PMI). The present invention encompasses amino acidvariations of the amino acid sequences disclosed in FIGS. 1A-1B and5A-5B and 6A-6B that have proteolytic activity. Such proteolytic aminoacid variants can be used in cleaning compositions.

[0021] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Asused herein “amino acid” refers to peptide or protein sequences orportions thereof. A “polynucleotide homolog” as used herein refers to agram-positive microorganism polynucleotide that has at least 80%, atleast 90% and at least 95% identity to B.subtilis CP1, CP2 or CP3, orwhich is capable of hybridizing to B.subtilis CP1, CP2 or CP3 underconditions of high stringency and which encodes an amino acid sequencehaving cysteine protease activity.

[0022] The terms “isolated” or “purified” as used herein refer to anucleic acid or amino acid that is removed from at least one componentwith which it is naturally associated.

[0023] As used herein, the term “heterologous protein” refers to aprotein or polypeptide that does not naturally occur in a gram-positivehost cell Examples of heterologous proteins include enzymes such ashydrolases including proteases, cellulases, amylases, carbohydrases, andlipases; isomerases such as racemases, epimerases, tautomerases, ormutases; transferases, kinases and phophatases. The heterologous genemay encode therapeutically significant proteins or peptides, such asgrowth factors, cytokines, ligands, receptors and inhibitors, as well asvaccines and antibodies. The gene may encode commercially importantindustrial proteins or peptides, such as proteases, carbohydrases suchas amylases and glucoamylases, cellulases, oxidases and lipases. Thegene of interest may be a naturally occurring gene, a mutated gene or asynthetic gene.

[0024] The term “homologous protein” refers to a protein or polypeptidenative or naturally occurring in a gram-positive host cell. Theinvention includes host cells producing the homologous protein viarecombinant DNA technology. The present invention encompasses agram-positive host cell having a deletion or interruption of naturallyoccurring nucleic acid encoding the homologous protein, such as aprotease, and having nucleic acid encoding the homologous protein, or avariant thereof, re-introduced in a recombinant form. In anotherembodiment, the host cell produces the homologous protein.

[0025] As used herein, the term “overexpressing” when refering to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

[0026] As used herein, the phrase “proteolytic activity” refers to aprotein that is able to hydrolyze a peptide bond. Enzymes havingproteolytic activity are described in Enzyme Nomenclature, 1992, editedWebb Academic Press, Inc.

[0027] Detailed Description of the Preferred Embodiments

[0028] The unexpected discovery of the cysteine proteases CP1, CP2 andCP3 in B.subtilis provides a basis for producing host cells, expressionmethods and systems which can be used to prevent the degradation ofrecombinantly produced heterologous proteins. In a preferred embodiment,the host cell is a gram-positive host cell that has a deletion ormutation in the naturally occurring cysteine protease said mutationresulting in deletion or inactivation of the production by the host cellof the proteolytic cysteine protease gene product. The host cell mayadditionally be genetically engineered to produced a desired protein orpolypeptide.

[0029] It may also be desired to genetically engineer host cells of anytype to produce a gram-positive cysteine protease. Such host cells areused in large scale fermentation to produce large quantities of thecysteine protease which may be isolated or purified and used in cleaningproducts, such as detergents.

[0030] I. Cysteine Protease Sequences

[0031] The CP1, CP2 and CP3 polynucleotides having the sequences asshown in FIGS. 1A-1B, 5A-5B and 6A-6B, respectively, encode the Bacillussubtilis cysteine proteases CP1, CP2 and CP3. As will be understood bythe skilled artisan, due to the degeneracy of the genetic code, avariety of polynucleotides can encode the Bacillus subtilis CP1, CP2 andCP3. The present invention encompasses all such polynucleotides.

[0032] The present invention encompasses CP1, CP2 and CP3 polynucleotidehomologs encoding gram-positive microorganism cysteine proteases CP1,CP2 and CP3, respectively, which have at least 80%, or at least 90% orat least 95% identity to B.subtilis CP1, CP2 and CP3 as long as thehomolog encodes a protein that has proteolytic activity.

[0033] Gram-positive polynucleotide homologs of B.subtilis CP1, CP2 orCP3 may be obtained by standard procedures known in the art from, forexample, cloned DNA (e.g., a DNA “library”). genomic DNA libraries, bychemical synthesis once identified, by cDNA cloning, or by the cloningof genomic DNA, or fragments thereof, purified from a desired cell.(See, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A PracticalApproach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred sourceis from genomic DNA. Nucleic acid sequences derived from genomic DNA maycontain regulatory regions in addition to coding regions. Whatever thesource, the isolated CP1, CP2 or CP3 gene should be molecularly clonedinto a suitable vector for propagation of the gene.

[0034] In the molecular cloning of the gene from genomic DNA, DNAfragments are generated, some of which will encode the desired gene. TheDNA may be cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

[0035] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the CP1, CP2 or CP3 may be accomplishedin a number of ways. For example, a B.subtilis CP1, CP2 or CP3 gene ofthe present invention or its specific RNA, or a fragment thereof, suchas a probe or primer, may be isolated and labeled and then used inhybridization assays to detect a gram-positive CP1, CP2 or CP3 gene.(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. AndHogness, D., 1975, Proc. Natl. Acad. Sci. USA 72:3961). Those DNAfragments sharing substantial sequence similarity to the probe willhybridize under stringent conditions.

[0036] Accordingly, the present invention provides a method for thedetection of gram-positive CP1, CP2 and CP3 polynucleotide homologswhich comprises hybridizing part or all of a nucleic acid sequence of B.subtilis CP1, CP2 and CP3 with gram-positive microorganism nucleic acidof either genomic or cDNA origin.

[0037] Also included within the scope of the present invention aregram-positive microorganism polynucleotide sequences that are capable ofhybridizing to the nucleotide sequence of B.subtilis CP1, CP2 or CP3under conditions of intermediate to maximal stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex, as taught in Berger and Kimmel (1987, Guide toMolecular Cloning Techniques. Methods in Enzymology, Vol 152, AcademicPress, San Diego Calif.) incorporated herein by reference, and confer adefined “stringency” as explained below.

[0038] “Maximum stringency” typically occurs at about Tm-5° C. (5° C.below the Tm of the probe); “high stringency” at about 5° C. to 10° C.below Tm; “intermediate stringency” at about 10° C. to 20° C. below Tm;and “low stringency” at about 20° C. to 25° C. below Tm. As willunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical polynucleotidesequences while an intermediate or low stringency hybridization can beused to identify or detect polynucleotide sequence homologs.

[0039] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs J (1994) Dictionary ofBiotechnology, Stockton Press, New York N.Y.).

[0040] The process of amplification as carried out in polymerase chainreaction (PCR) technologies is described in Dieffenbach C W and G SDveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring HarborPress, Plainview N.Y.). A nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides from B. subtilis CP1,CP2 or CP3 preferably about 12 to 30 nucleotides, and more preferablyabout 20-25 nucleotides can be used as a probe or PCR primer.

[0041] The B.subtilis amino acid sequences CP1, CP2 and CP3 (shown inFIGS. 2, 4 and 3, respectively) were identified via a FASTA search ofBacillus subtilis genomic nucleic acid sequences. B. subtilis CP1 (YJDE)was identified by its structural homology to the cysteine proteasepapain having the sequence designated “papa_carpa.p”. As shown in FIG.2, YJDE has the motif GXCWAF as well as the conserved catalytic residuesHis/Ala and Asn/Ser. CP2 (YdHS) and CP3 (PMI) were identified upon theirstructural homology to CP1 (YJDE). The presence of GXCWAF as well asresidues His/Ala and Asn/Ser is noted in FIGS. 3 and 4. CP3 (PMI) waspreviously characterized as a possible phosphomannose isomerase,(Noramata). There has been no previous characterization of CP3 as acysteine protease.

[0042] II. Expression Systems

[0043] The present invention provides host cells, expression methods andsystems for the enhanced production and secretion of desiredheterologous or homologous proteins in gram-positive microorganisms. Inone embodiment, a host cell is genetically engineered to have a deletionor mutation in the gene encoding a gram-positive CP1, CP2 or CP3 suchthat the respective activity is deleted. In another embodiment of thepresent invention, a gram-positive microorganism is geneticallyengineered to produce a cysteine protease of the present invention.

[0044] Inactivation of a Gram-Positive Cysteine Protease in a Host Cell

[0045] Producing an expression host cell incapable of producing thenaturally occurring cysteine protease necessitates the replacementand/or inactivation of the naturally occurring gene from the genome ofthe host cell. In a preferred embodiment, the mutation is anon-reverting mutation.

[0046] One method for mutating nucleic acid encoding a gram-positivecysteine protease is to clone the nucleic acid or part thereof, modifythe nucleic acid by site directed mutagenesis and reintroduce themutated nucleic acid into the cell on a plasmid. By homologousrecombination, the mutated gene may be introduced into the chromosome.In the parent host cell, the result is that the naturally occurringnucleic acid and the mutated nucleic acid are located in tandem on thechromosome. After a second recombination, the modified sequence is leftin the chromosome having thereby effectively introduced the mutationinto the chromosomal gene for progeny of the parent host cell.

[0047] Another method for inactivating the cysteine protease proteolyticactivity is through deleting the chromosomal gene copy. In a preferredembodiment, the entire gene is deleted, the deletion occurring in suchas way as to make reversion impossible. In another preferred embodiment,a partial deletion is produced, provided that the nucleic acid sequenceleft in the chromosome is too short for homologous recombination with aplasmid encoded cysteine protease gene. In another preferred embodiment,nucleic acid encoding the catalytic amino acid residues are deleted.

[0048] Deletion of the naturally occurring gram-positive microorganismcysteine protease can be carried out as follows. A cysteine proteasegene including its 5′ and 3′ regions is isolated and inserted into acloning vector. The coding region of the cysteine protease gene isdeleted form the vector in vitro, leaving behind a sufficient amount ofthe 5′ and 3′ flanking sequences to provide for homologous recombinationwith the naturally occurring gene in the parent host cell. The vector isthen transformed into the gram-positive host cell. The vector integratesinto the chromosome via homologous recombination in the flankingregions. This method leads to a gram-positive strain in which theprotease gene has been deleted.

[0049] The vector used in an integration method is preferably a plasmid.A selectable marker may be included to allow for ease of identificationof desired recombinant microorgansims. Additionally, as will beappreciated by one of skill in the art, the vector is preferably onewhich can be selectively integrated into the chromosome. This can beachieved by introducing an inducible origin of replication, for example,a temperature sensitive origin into the plasmid. By growing thetransformants at a temperature to which the origin of replication issensitive, the replication function of the plasmid is inactivated,thereby providing a means for selection of chromosomal integrants.Integrants may be selected for growth at high temperatures in thepresence of the selectable marker, such as an antibiotic. Integrationmechanisms are described in WO 88/06623.

[0050] Integration by the Campbell-type mechanism can take place in the5′ flanking region of the protease gene, resulting in a proteasepositive strain carrying the entire plasmid vector in the chromosome inthe cysteine protease locus. Since illegitimate recombination will givedifferent results it will be necessary to determine whether the completegene has been deleted, such as through nucleic acid sequencing orrestriction maps.

[0051] Another method of inactivating the naturally occurring cysteineprotease gene is to mutagenize the chromosomal gene copy by transforminga gram-positive microorganism with oligonucleotides which are mutagenic.Alternatively, the chromosomal cysteine protease gene can be replacedwith a mutant gene by homologous recombination.

[0052] The present invention encompasses host cells having deletions ormutations of a cysteine protease of the present invention as well asadditional protease deletions or mutations, such as deletions ormutations in apr, npr, epr, mpr and others known to those of skill inthe art. U.S. Pat. No. 5,264,366 discloses Bacillus host cells having adeletion of apr and npr; U.S. Pat. No. 5,585,253 discloses Bacillus hostcells having a deletion of epr; Margot et al., 1996, Microbiology 142:3437-3444 disclose host cells having a deletion in wpr and EP patent0369817 discloses Bacillus host cells having a deletion of mpr.

[0053] One assay for the detection of mutants involves growing theBacillus host cell on medium containing a protease substrate andmeasuring the appearance or lack thereof, of a zone of clearing or haloaround the colonies. Host cells which have an inactive protease willexhibit little or no halo around the colonies.

[0054] III. Production of Cysteine Protease

[0055] For production of cysteine protease in a host cell, an expressionvector comprising at least one copy of nucleic acid encoding agram-positive microorganism CP1, CP2 or CP3, and preferably comprisingmultiple copies, is transformed into the host cell under conditionssuitable for expression of the cysteine protease. In accordance with thepresent invention, polynucleotides which encode a gram-positivemicroorganism CP1, CP2 or CP3, or fragments thereof, or fusion proteinsor polynucleotide homolog sequences that encode amino acid variants ofB.subtilis CP1, CP2 or CP3, may be used to generate recombinant DNAmolecules that direct their expression in host cells. In a preferredembodiment, the gram-positive host cell belongs to the genus Bacillus.In another preferred embodiment, the gram positive host cell is B.subtilis.

[0056] As will be understood by those of skill in the art, it may beadvantageous to produce polynucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulargram-positive host cell (Murray E et al (1989) Nuc Acids Res 17:477-508)can be selected, for example, to increase the rate of expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

[0057] Altered CP1, CP2 or CP3 polynucleotide sequences which may beused in accordance with the invention include deletions, insertions orsubstitutions of different nucleotide residues resulting in apolynucleotide that encodes the same or a functionally equivalent CP1,CP2 or CP3 homolog, respectively. As used herein a “deletion” is definedas a change in either nucleotide or amino acid sequence in which one ormore nucleotides or amino acid residues, respectively, are absent.

[0058] As used herein an “insertion” or “addition” is that change in anucleotide or amino acid sequence which has resulted in the addition ofone or more nucleotides or amino acid residues, respectively, ascompared to the naturally occurring CP1, CP3 or CP3.

[0059] As used herein “substitution” results from the replacement of oneor more nucleotides or amino acids by different nucleotides or aminoacids, respectively.

[0060] The encoded protein may also show deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally CP1, CP2 or CP3 variant. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues as long as the variant retains theability to modulate secretion. For example, negatively charged aminoacids include aspartic acid and glutamic acid: positively charged aminoacids include lysine and arginine; and amino acids with uncharged polarhead groups having similar hydrophilicity values include leucine,isoleucine, valine; glycine, alanine; asparagine, glutamine; serine,threonine, phenylalanine, and tyrosine.

[0061] The CP1, CP2 or CP3 polynucleotides of the present invention maybe engineered in order to modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques which are well known in the art, eg, site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns or to change codon preference, for example.

[0062] In one embodiment of the present invention, a gram-positivemicroorganism CP1, CP2 or CP3 polynucleotide may be ligated to aheterologous sequence to encode a fusion protein. A fusion protein mayalso be engineered to contain a cleavage site located between thecysteine protease nucleotide sequence and the heterologous proteinsequence, so that the cysteine protease may be cleaved and purified awayfrom the heterologous moiety.

[0063] IV. Vector Sequences

[0064] Expression vectors used in expressing the cysteine proteases ofthe present invention in gram-positive microorganisms comprise at leastone promoter associated with a cysteine protease selected from the groupconsisting of CP1, CP2 and CP3, which promoter is functional in the hostcell. In one embodiment of the present invention, the promoter is thewild-type promoter for the selected cysteine protease and in anotherembodiment of the present invention, the promoter is heterologous to thecysteine protease, but still functional in the host cell. In onepreferred embodiment of the present invention, nucleic acid encoding thecysteine protease is stably integrated into the microorganism genome.

[0065] In a preferred embodiment, the expression vector contains amultiple cloning site cassette which preferably comprises at least onerestriction endonuclease site unique to the vector, to facilitate easeof nucleic acid manipulation. In a preferred embodiment, the vector alsocomprises one or more selectable markers. As used herein, the termselectable marker refers to a gene capable of expression in thegram-positive host which allows for ease of selection of those hostscontaining the vector. Examples of such selectable markers include butare not limited to antibiotics, such as, erythromycin, actinomycin,chloramphenicol and tetracycline.

[0066] V. Transformation

[0067] A variety of host cells can be used for the production of CP1,CP2 and CP3 including bacterial, fungal, mammalian and insects cells.General transformation procedures are taught in Current Protocols InMolecular Biology (vol.1, edited by Ausubel et al., John Wiley & Sons,Inc. 1987, Chapter 9) and include calcium phosphate methods,transformation using DEAE-Dextran and electroporation. Planttransformation methods are taught in Rodriquez (WO 95/14099, publishedMay 26, 1995).

[0068] In a preferred embodiment, the host cell is a gram-positivemicroorganism and in another preferred embodiment, the host cell isBacillus. In one embodiment of the present invention, nucleic acidencoding one or more cysteine protease(s) of the present invention isintroduced into a host cell via an expression vector capable ofreplicating within the Bacillus host cell. Suitable replicating plasmidsfor Bacillus are described in Molecular Biological Methods for Bacillus,Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expresslyincorporated by reference; see chapter 3 on plasmids. Suitablereplicating plasmids for B. subtilis are listed on page 92.

[0069] In another embodiment, nucleic acid encoding a cysteineprotease(s) of the present invention is stably integrated into themicroorganism genome. Preferred host cells are gram-positive host cells.Another preferred host is Bacillus. Another preferred host is Bacillussubtilis. Several strategies have been described in the literature forthe direct cloning of DNA in Bacillus. Plasmid marker rescuetransformation involves the uptake of a donor plasmid by competent cellscarrying a partially homologous resident plasmid (Contente et al.,Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191(1990); Weinrauch et al., J. Bacteriol. 154(3):1077-1087 (1983); andWeinrauch et al., J. Bacteriol. 169(3):1205-1211 (1987)). The incomingdonor plasmid recombines with the homologous region of the resident“helper” plasmid in a process that mimics chromosomal transformation.

[0070] Transformation by protoplast transformation is described for B.subtilis in Chang and Cohen, (1979) Mol. Gen. Genet 168:111-115; forB.megaterium in Vorobjeva et al., (1980) FEMS Microbiol. Letters7:261-263; for B. amyloliquefaciens in Smith et al., (1986) Appl. andEnv. Microbiol. 51:634; for B.thuringiensis in Fisher et al., (1981)Arch. Microbiol. 139:213-217; for B.sphaericus in McDonald (1984) J.Gen. Microbiol. 130:203; and B.larvae in Bakhiet et al., (1985) 49:577.Mann et al., (1986, Current Microbiol. 13:131-135) report ontransformation of Bacillus protoplasts and Holubova, (1985) FoliaMicrobiol. 30:97) disclose methods for introducing DNA into protoplastsusing DNA containing liposomes.

[0071] VI. Identification of Transformants

[0072] Whether a host cell has been transformed with a mutated or anaturally occurring gene encoding a gram-positive CP1, CP2 or CP3,detection of the presence/absence of marker gene expression can suggestswhether the gene of interest is present However, its expression shouldbe confirmed. For example, if the nucleic acid encoding a cysteineprotease is inserted within a marker gene sequence, recombinant cellscontaining the insert can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem withnucleic acid encoding the cysteine protease under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the cysteine protease aswell.

[0073] Alternatively, host cells which contain the coding sequence for acysteine protease and express the protein may be identified by a varietyof procedures known to those of skill in the art. These proceduresinclude, but are not limited to, DNA-DNA or DNA-RNA hybridization andprotein bioassay or immunoassay techniques which include membrane-based,solution-based, or chip-based technologies for the detection and/orquantification of the nucleic acid or protein.

[0074] The presence of the cysteine polynucleotide sequence can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes, portions or fragments of B.subtilis CP1, CP2 or CP3.

[0075] VII. Assay of Protease Activity

[0076] There are various assays known to those of skill in the art fordetecting and measuring protease activity. There are assays based uponthe release of acid-soluble peptides from casein or hemoglobin measuredas absorbance at 280 nm or colorimetrically using the Folin method(Bergmeyer, et al., 1984, Methods of Enzymatic Analysis vol. 5,Peptidases, Proteinases and their Inhibitors, Verlag Chemie, Weinheim).Other assays involve the solubilization of chromogenic substrates (Ward,1983, Proteinases, in Microbial Enzymes and Biotechnology (W. M.Fogarty, ed.), Applied Science, London, pp. 251-317).

[0077] VIII. Secretion of Recombinant Proteins

[0078] Means for determining the levels of secretion of a heterologousor homologous protein in a gram-positive host cell and detectingsecreted proteins include, using either polyclonal or monoclonalantibodies specific for the protein. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton R et al (1990, Serological Methods, aLaboratory Manual, APS Press, St Paul Minn.) and Maddox Del. et al(1983, J Exp Med 158:1211).

[0079] A wide variety of labels and conjugation techniques are known bythose skilled in the art and can be used in various nucleic and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligolabeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

[0080] A number of companies such as Pharmacia Biotech (PiscatawayN.J.), Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio)supply commercial kits and protocols for these procedures. Suitablereporter molecules or labels include those radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567 and incorporated herein by reference.

[0081] IX. Purification of Proteins

[0082] Gram positive host cells transformed with polynucleotidesequences encoding heterologous or homologous protein may be culturedunder conditions suitable for the expression and recovery of the encodedprotein from cell culture. The protein produced by a recombinantgram-positive host cell comprising a mutation or deletion of thecysteine protease activity will be secreted into the culture media.Other recombinant constructions may join the heterologous or homologouspolynucleotide sequences to nucleotide sequence encoding a polypeptidedomain which will facilitate purification of soluble proteins (Kroll D Jet al (1993) DNA Cell Biol 12:441-53).

[0083] Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath J (1992)Protein Expr Purif 3:263-281), protein A domains that allow purificationon immobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp, Seattle Wash.).The inclusion of a cleavable linker sequence such as Factor XA orenterokinase (Invitrogen, San Diego Calif.) between the purificationdomain and the heterologous protein can be used to facilitatepurification.

[0084] X. Uses of The Present Invention

[0085] CP1, CP2 and CP3 and Genetically Engineered Host Cells

[0086] The present invention provides genetically engineered host cellscomprising preferably non-revertable mutations or deletions in thenaturally occurring gene encoding CP1, CP2 or CP3 such that theproteolytic activity is diminished or deleted altogether. The host cellmay contain additional protease deletions, such as deletions of themature subtilisn protease and/or mature neutral protease disclosed inU.S. Pat. No. 5,264,366.

[0087] In a preferred embodiment, the host cell is further geneticallyengineered to produce a desired protein or polypeptide. In a preferredembodiment the host cell is a Bacillus. In another preferred embodiment,the host cell is a Bacillus subtilis.

[0088] In an alternative embodiment, a host cell is geneticallyengineered to produce a gram-positive CP1, CP2 or CP3. In a preferredembodiment, the host cell is grown under large scale fermentationconditions, the CP1, CP2 or CP3 is isolated and/or purified and used incleaning compositions such as detergents. Detergent formulations aredisclosed in WO 95/10615. A cysteine protease of the present inventioncan be useful in formulating various cleaning compositions. A number ofknown compounds are suitable surfactants useful in compositionscomprising the cysteine protease of the invention. These includenonionic, anionic, cationic, anionic or zwitterionic detergents, asdisclosed in U.S. Pat. Nos. 4,404,128 and 4,261,868. A suitabledetergent formulation is that described in Example 7 of U.S. Pat. No.5,204,015. The art is familiar with the different formulations which canbe used as cleaning compositions. In addition, a cysteine protease ofthe present invention can be used, for example, in bar or liquid soapapplications, dishcare formulations, contact lens cleaning solutions orproducts, peptide hydrolysis, waste treatment, textile applications, asfusion-cleavage enzymes in protein production, etc. A cysteine proteasemay provide enhanced performance in a detergent composition (as comparedto another detergent protease). As used herein, enhanced performance ina detergent is defined as increasing cleaning of certain enzymesensitive stains such as grass or blood, as determined by usualevaluation after a standard wash cycle.

[0089] A cysteine protease of the present invention can be formulatedinto known powdered and liquid detergents having pH between 6.5 and 12.0at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight.These detergent cleaning compositions can also include other enzymessuch as known proteases, amylases, cellulases, lipases orendoglycosidases, as well as builders and stabilizers.

[0090] The addition of a cysteine protease to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe present compositions as long as the pH is within the above range,and the temperature is below the described cysteine protease denaturingtemperature. In addition, a cysteine protease can be used in a cleaningcomposition without detergents, again either alone or in combinationwith builders and stabilizers.

[0091] One aspect of the invention is a composition for the treatment ofa textile that includes a cysteine protease of the present invention.The composition can be used to treat for example silk or wool asdescribed in publications such as RD 216,034; EP 134,267; U.S. Pat No.4,533,359; and EP 344,259.

[0092] Proteases can be included in animal feed such as part of animalfeed additives as described in, for example, U.S. Pat. Nos. 5,612,055;5,314,692; and 5,147,642.

[0093] CP1, CP2 and CP3 Polynucleotides

[0094] A B.subtlis polynucleotide, or any part thereof, provides thebasis for detecting the presence of gram-positive microorganismpolynucleotide homologs through hybridization techniques and PCRtechnology.

[0095] Accordingly, one aspect of the present invention is to providefor nucleic acid hybridization and PCR probes which can be used todetect polynucleotide sequences, including genomic and cDNA sequences,encoding gram-positive CP1, CP2 or CP3 or portions thereof.

[0096] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto

EXAMPLE I Preparation of a Genomic Library

[0097] The following example illustrates the preparation of a Bacillusgenomic library.

[0098] Genomic DNA from Bacillus cells is prepared as taught in CurrentProtocols In Molecular Biology vol. 1, edited by Ausubel et al., JohnWiley & Sons, Inc. 1987, chapter 2. 4.1. Generally, Bacillus cells froma saturated liquid culture are lysed and the proteins removed bydigestion with proteinase K. Cell wall debris, polysaccharides, andremaining proteins are removed by selective precipitation with CTAB, andhigh molecular weight genomic DNA is recovered from the resultingsupernatant by isopropanol precipitation. If exceptionally clean genomicDNA is desired, an additional step of purifying the Bacillus genomic DNAon a cesium chloride gradient is added.

[0099] After obtaining purified genomic DNA, the DNA is subjected toSau3A digestion. Sau3A recognizes the 4 base pair site GATC andgenerates fragments compatible with several convenient phage lambda andcosmid vectors. The DNA is subjected to partial digestion to increasethe chance of obtaining random fragments.

[0100] The partially digested Bacillus genomic DNA is subjected to sizefractionation on a 1% agarose gel prior to cloning into a vector.Alternatively, size fractionation on a sucrose gradient can be used. Thegenomic DNA obtained from the size fractionation step is purified awayfrom the agarose and ligated into a cloning vector appropriate for usein a host cell and transformed into the host cell.

EXAMPLE II Detection of Gram-Postive Microorganisms

[0101] The following example describes the detection of gram-positivemicroorganism CP1. The same procedures can be used to detect CP2 andCP3.

[0102] DNA derived from a gram-positive microorganism is preparedaccording to the methods disclosed in Current Protocols in MolecularBiology, Chap. 2 or 3. The nucleic acid is subjected to hybridizationand/or PCR amplification with a probe or primer derived from CP1. Apreferred probe comprises the nucleic acid section containing theconserved motif GXCWAF.

[0103] The nucleic acid probe is labeled by combining 50 pmol of thenucleic acid and 250 mCi of [gamma ³²P] adenosine triphosphate(Amersham, Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston Mass.). The labeled probe is purified with Sephadex G-25 superfine resin column (Pharmacia). A portion containing 10⁷ counts perminute of each is used in a typical membrane based hybridizationanalysis of nucleic acid sample of either genomic or cDNA origin.

[0104] The DNA sample which has been subjected to restrictionendonuclease digestion is fractionated on a 0.7 percent agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40 degrees C.To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. The blots are exposed tofilm for several hours, the film developed and hybridization patternsare compared visually to detect polynucleotide homologs of B.subtilisCP1. The homologs are subjected to confirmatory nucleic acid sequencing.Methods for nucleic acid sequencing are well known in the art.Conventional enzymatic methods employ DNA polymerase Klenow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest.

[0105] Various other examples and modifications of the foregoingdescription and examples will be apparent to a person skilled in the artafter reading the disclosure without departing from the spirit and scopeof the invention, and it is intended that all such examples orodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1. A gram-positive microorganism having a mutation or deletion of partor all of the gene encoding CP1 said mutation or deletion resulting inthe inactivation of the CP1 proteolytic activity.
 2. A gram-positivemicroorganism having a mutation or deletion of part or all of the geneencoding CP2 said mutation or deletion resulting in the inactivation ofthe CP2 proteolytic activity.
 3. A gram-positive microorganism having amutation or deletion of part or all of the gene encoding CP3 saidmutation or deletion resulting in the inactivation of the CP3proteolytic activity.
 4. The gram-positive microorganism according toclaims 1, 2 or 3 that is a member of the family Bacillus.
 5. Themicroorganism according to claim 4 wherein the member is selected fromthe group consisting of B. subtilis, B. licheniformis, B. lentus, B.brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
 6. Themicroorganism of claim 1, 2 or 3 wherein said microorganism is capableof expressing a heterologous protein.
 7. The microorganism of claim 6wherein said heterologous protein is selected from the group consistingof hormone, enzyme, growth factor and cytokine.
 8. The microorganism ofclaim 7 wherein said heterologous protein is an enzyme.
 9. Themicroorganism of claim 8 wherein said enzyme is selected from the groupconsisting of a proteases, carbohydrases, and lipases; isomerases suchas racemases, epimerases, tautomerases, or mutases; transferases,kinases and phophatases.
 10. A cleaning composition comprising at leastone cysteine protease selected from the group consisting of CP1, CP2 andCP3.
 11. An expression vector comprising nucleic acid encoding acysteine protease selected from the group consisting of CP1, CP2 andCP3.
 12. A host cell comprising an expression vector according to claim11.
 13. A method for the production of a heterologous protein in aBacillus host cell comprising the steps of (a) obtaining a Bacillus hostcell comprising nucleic acid encoding said heterologous protein whereinsaid host cell contains a mutation or deletion in at least one of thegenes encoding cysteine protease 1, cysteine protease 2 and cysteineprotease 3; and (b) growing said Bacillus host cell under conditionssuitable for the expression of said heterologous protein.
 14. The methodof claim 13 wherein said Bacillus cell is selected from the groupconsisting of Bacillus subtilis, B. licheniformis, B. lentus, B. brevis,B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B.coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
 15. Themethod of claim 13 wherein said Bacillus host cell further comprises amutation or deletion in at least one of the genes encoding apr, npr,epr, wpr and mrp.
 16. A gram-positive microorganism having at mutationor deletion in at least one of the genes encoding a cysteine proteaseselected from the group consisting of CP1, CP2 and CP3.
 17. Themicroorganism of claim 16 further comprising a mutation or deletion inat least one of the genes encoding apr, npr, epr, wpr and mrp.